Universal method and composition for the rapid lysis of cells for the release of nucleic acids and their detection

ABSTRACT

This invention describes a rapid (10 to 15 minutes), simple, flexible and efficient method of nucleic acids extraction for nucleic acid testing assays. This method has the following basic steps: i) mechanical cell lysis using solid particles in the presence of a chelating agent, followed by ii) controlling the presence and/or activity of NAT assays inhibitors. This method is applicable to various biological samples and universal for microorganisms, as one can use it to extract nucleic acids from test samples containing target viruses, bacteria, bacterial spores, fungi, parasites or other eukaryotic cells, including animal and human cells.

BACKGROUND OF THE INVENTION Classical Methods for the Isolation ofNucleic Acids from Microorganisms

[0001] With the advent of molecular biology, an increasing number ofdiagnostic methods are based on the detection of nucleic acids. Nucleicacid amplification technologies represent useful tools in molecularbiology. Since the discovery of the polymerase chain reaction (PCR),various protocols have been described for isolating nucleic acidssuitable for detection and identification of microorganisms. However,most of these protocols are time-consuming and often require the use oftoxic chemicals. In addition, protocols need to be tailor-made for eachmicrobe type; a lysis protocol for fungi may not be suitable forgram-negative bacteria, or parasites, or bacterial spores, and so on.Furthermore, these protocols require numerous steps, increasing the riskof sample-to-sample or carry-over contamination.

[0002] Hugues et al. (Methods in Microbiology, 1971, vol. 5B, AcademicPress, New York) have reviewed the available methods for disintegratingmicrobes for preparing biologically active fractions. The methodselected will depend on its capability to process samples of a certainsize or to be able to process multiple samples in a reasonable period oftime, while the desired nucleic acids retain their integrity. Classicalphysical methods of cell breakage include mechanical cell disintegration(crushing and grinding, wet milling, ultrasonics, hydraulic shear,freeze pressure), liquid or hydrodynamic shear (French press, Chaikoffpress, homogenizers, wet mills, vibration mills, filters, ultrasonicdisintegration) and solid shear (grinding, Hugues press). Chemicalmethods of cell disintegration are mostly aimed at modifying the cellwall or cytoplasmic membrane, or both, so that the cells either becomeleaky or burst due to the effects of turgor pressure. Methods includeosmosis, drying and extraction, autolysis, inhibition of cell wallsynthesis, enzymic attack on cell walls, bacteriophages and other lyticfactors, and ionizing radiation.

[0003] Several methods of cell disruption, with or without solidparticles, and involving physical agitation to release nucleic acidshave been described. Examples include a cell disrupter from Amoco (U.S.Pat. No. 5,464,773) and the FastPrep® apparatus from Qbiogene (U.S. Pat.No. 5,567,050). However, no examples of reduction into practice wereprovided in those patents. Other commercial devices that may be used tolyse cells using similar shaking-type bead mills include theMicro-Dismembrator II from B. Braun Biotech (Allentown, Pa., USA) andthe Mini-BeadBeater from BioSpec (Bartlesville, Okla., USA). However,most methods described in the literature using this type of apparatusrequire additional nucleic acid purification steps after the lysis step(Miller et al., 1999, Appl. Env. Microbiol. 65: 4715-4724; Cornejo etal., 1998, Appl. Env. Microbiol. 64: 3099-3101). In addition, mostprotocols rely on the presence of lysogenic chemicals to enhancemechanical cell lysis. A major drawback of these methods is that thechemicals used often adversely affect subsequent molecular biologyprocesses, such as the detergent sodium dodecyl sulfate (SDS) in PCR.

[0004] Significant progress has been made in the last few years bymanufacturers to improve the simplicity of release and purification ofnucleic acids from microorganisms present in clinical specimens.However, for laboratory personnel, it is not obvious to make a choice inthe plethora of commercially available nucleic acids extraction kits.Those kits exhibit variable performances in the preparation of varioustest samples containing diverse target microorganisms for nucleic acidstesting (NAT). These kits have different total nucleic acids recovery,number of manipulation steps and time requirements. Example 2 shows acomparison of 8 different commercially available DNA extraction kitswith the method described in this invention. The conclusion was thatwith regards to speed, total recovery of DNA and number of manipulationsteps, the sample preparation method described below was the bestprotocol.

A Novel Extraction M thod that is Simpl , Rapid, Universal and Versatil

[0005] The method described in this invention is a Rapid Universal CellLysis and Nucleic Acids Preparation (RUCLANAP) protocol. It requiresapproximately 10 to 15 min, in a simple, flexible and efficientprotocol. The main sequence of events underlying this invention are aninitial mechanical cell lysis using solid particles in the presence of achelating agent, followed by a control of substances inhibitory topolymerases as used in amplification reactions, or inhibitory to othersteps of NAT assays. Said inhibitory substances are present in the testsample and/or are released upon cell lysis into the lysate. For mostapplications, no further purification of nucleic acids is required. Sucha control can be effected by diverse ways such as heating, adsorbing,freezing, removing or diluting the NAT inhibitors in the sample or thelysate. The advantages of this method are simplicity, rapidity,efficiency, universality (effective with all microbial species) and lowcost. Prior to the present invention, heat was used to lyse cells (U.S.Pat. No. 5,376,527), agitation with particles was used to lyse cells(U.S. Pat. No. 5,567,050 and 4,295,613, and World Pat. No. WO 02/10333),agitation with particles in organic solvents was used to lyse cells(U.S. Pat. No. 5,643,767), and agitation with particles was applied toalready heat-lysed cells to provide access to nucleic acids (U.S. Pat.No. 5,942,425). Prior to the present invention, the particular sequenceof events (agitation with particles in chelating buffer followed by heatinactivation of PCR inhibitors) was applied to prepare DNA for molecularbiology. However, in these earlier methods, cell lysis did not rely uponmechanical forces, but mainly on heat provided by a boiling step. Forexample, others have used the combination of a vortexing step inChelex®, a weak ion-exchange matrix, followed by boiling of the sample(Kessler et al., 1997, J. Clin. Microbiol. 35: 1592-1594; Drake et al.,1996, Food Res. Int. 29: 451-455; Yoshimi et al., 1993, Acta Pathol.Jap. 43: 790-791). In all cases, the very short vortex step wasperformed only to mix the Chelex® with the sample in order to sequesterdivalent cations, and bind compounds which inhibit PCR (Singer-Sam etal., 1989, Amplifications 3: 11); while cell lysis was obtained duringthe heating step, a process that is not universal for all microbes,especially bacterial spores and yeasts cells. As revealed in thisinvention in Example 19, the heating steps during the PCR protocol arenot sufficient to lyse Mycobacterium smegmatis.

[0006] Patent publication WO 99/15621 describes a method formechanically lysing bacteria or yeasts wherein a liquid samplecomprising the bacteria or yeasts is placed in a container withparticles having 90-150 μm, namely 100 μm for bacteria and the largeones having 400-600 μm, namely 500 μm for yeasts. This method is notoptimal, since after 8 minutes of vortex, about 60% of S. epidermidisand C. albicans are lysed.

[0007] Patent publication WO 00/05338 discloses a <<magnetic>> vortexmethod for lysing cells. At least two sizes of particles are used tomechanically break the cells. The larger particles are composed of amaterial which respond to a magnetic field (iron, namely), while thesmaller ones are non-magnetic beads. The large beads are placed inmovement by actuating a magnetic device and the sample is crushedbetween the large and the small beads, the smaller being moved by thelarger ones. The ratio of size between the small beads and the targetcells is rather small (50/1) while the ratio of size between the largeand small beads is relatively large (40/1). The large beads havetherefore the function of crushing the target cells between them and thesmall beads to free the cell contents into the lysate solution. Further,this application only provides a <<qualitative>> appreciation of thelysis rate and of the integrity of the nucleic acids obtained therefrom.Only electrophoretic gels show the results of the process. Amplificationprocess further performed on nucleic acids are very sensitive processeswhich require a high level of lysis liability and reproducibility. WO00/05338 is totally silent on achieving this high standard with thedisclosed <<magnetic>> vortex method.

[0008] Patent publication WO 02/10333 describes a method which may beadapted for different techniques: sonication, mechanical vortex andmagnetic vortex. The method comprises at least three parameters selectedfrom: a) 50-100% of particles mass with regards to the total mass of thesample to be treated, b) a ratio of particles of a small diameter toparticles of a large diameter (when two sizes of particles are used)which is less than or equal to 50%, c) a lysis duration of 9 to 20minutes, d) glass beads that are involved in the motion of the particlesand not in the lysis per se which are in a number of less than 7; and e)iron beads that have the same role as in d) but for a magnetic vortex,which are in a number of 5 to 15. Again the lysing particles have thesize as in WO 99/15621 (the small ones having 90-150 μm, namely 100 μmfor bacteria and the large ones having 400-600 μm, namely 500 μm foryeasts). Larger particle size ratios are favoured when a mixture ofparticles sizes is used (more than 50%), along with a relatively longlysis duration. Except for sonication, the other agitation techniquesrequire large non-lysing particles which are present to put the smallparticles into motion. These big particles may render the bead matrixmore voluminous and bumpy at the interface with the lysate, increasingthe dead volume, and hence rendering the recovery by pipetting of thelatter more difficult.

[0009] A publication by Jaffe et aL. (J. Clin. Microbiol., 2000, 38:3407-3412) discloses the use of 1.0 g of 100 μm glass beads and 0.25 gof Chelex-100 in a volume of 0.5 ml of bacterial sample to mechanicallybreak cells and release nucleic acids, further followed by a heatingstep. The process which is called “Bead beating with Chelex” howeverlacks sensitivity towards certain species, namely methicillin-resistantS. aureus (MRSA) even if the sensitivity is acceptable for otherbacteria easier to break up, such as gram-negative species. There is nosuggestion in this reference to vary the size of beads to improve thesensitivity and the versatility (“universality”) of the method forrecovering efficaciously nucleic acids from any microoganism.Furthermore, this method has not been tested with clinical samples.

[0010] During the 99^(th) ASM General Meeting held in Chicago in 1999,(Abstract C-481), the present inventors have disclosed the results of acomparative study conducted with the present method and kit (“thenreferred to as the IDI DNA extraction kit”) with eight other kits (seeExample 2). No details were given on the protocol for lysing the cellsand the components involved therein.

[0011] The RUCLANAP method is universal for microorganisms, as one canuse it to extract nucleic acids from viruses, bacteria, bacterialspores, fungi, parasites or from other eukaryotic cells, includinganimal and human cells. The basic RUCLANAP protocol is versatile, as itcan easily be adapted, depending on the type of clinical sample used, inorder to dilute or concentrate the microorganisms present in the sample,and/or to extract crude nucleic acids, and/or to control (inactivate,adsorb, dilute or remove) sample-specific inhibitors of NAT assays(Wilson, Applied Env. Microbiol., 1997, 63:3741-3751). For geneticanalysis of RNA, RNase inhibitors may be added to the sample beforenucleic acid extraction with RUCLANAP; no additional purification oftotal RNA is necessary for subsequent amplification and detection, suchas with RT-PCR (see Example 18).

[0012] The applications for this method are numerous: the samples fromwhich microbial nucleic acids have been successfully prepared using thismethod include microbial broth cultures, positive blood cultures,suspensions of microbial colonies grown on agar media, as well as avariety of biological samples including blood, plasma, plateletconcentrates, urine, cerebrospinal fluid, amniotic fluid, meconium,wound exudate, stools, nasal swabs, throat swabs, anal swabs, vaginalswabs, vaginal/anal swabs, rectal swabs, and bovine milk. The efficiencyof Streptococcus agalactiae lysis by the RUCLANAP method was estimatedto 99.99%, as determined by viable counts (see Example 5). For detailedsample preparation protocols with various specimens, see Examples 2 to12, 15, and 17-20. The method is also amendable to scaling up or down,and automation.

[0013] One concern in the handling of biological specimens is safety.Heating of a clinical sample containing infectious agents is one of thepreferred methods to render samples safer for handling. U.S. Pat. No.5,376,527 describes a lysis-effective amount of heat that is sufficientto render difficult-to-lyse cells of Mycobacterium tuberculosisnon-infectious. We have found that the combination of the cell lysisstep and the heating step of the RUCLANAP method also renders mostclinical samples safer for handling. A heating and/or freezing step maybe performed prior to RUCLANAP to render test samples safer forhandling.

SUMMARY OF THE INVENTION

[0014] It is an object of the present invention to provide a universalmethod and composition or kit for the rapid preparation of nucleic acidsfrom cells, namely microbial cells or viruses, said method comprising:

[0015] A. Placing biological samples in the presence of a granulometricgradient formed by solid particles having nominal diameter sizescomprised between 75 μm and 200 μm;

[0016] B. Submitting said mixture to sufficient mechanical forces todisrupt cell walls and membranes, to release cellular and sub-cellular(mitochondria) contents further submitted to a step of controlling thepresence and/or activity of inhibitors of NAT assays.

[0017] Namely, the particles have nominal diameter sizes higher than 100μm and lower than 1500 μm. In a preferred embodiment, the particlesessentially consist of a nominal diameter size varying from 150 to about1200 μm, namely more than 150 μm and lower than or equal to 1180 μm.

[0018] In a further preferred embodiment, the solid spherical particlesare acid-washed glass beads of diameter of two ranges: one from 150 to212 μm, and another one from 710 to 1180 μm. For universal cell(bacteria, yeasts, fungi, parasites, animal and human cells) or viruslysis, the beads are preferably mixed in a 4:1 ratio (w/w), and there isa total of 0.05 g of glass bead matrix per 1.5 mL microtube. Other beadratios are also possible; for example, to lyse yeast cells, the beadsare preferably mixed in a 1:1 ratio (w/w) or higher, and there is about0.02 g of glass bead matrix per 1.5 mL microtube. When virus particlesare also a target for lysis, a third range of about 75 to 106 μm may beadded, if necessary or desirable.

[0019] In another preferred embodiment, the biological sample andparticles are in a buffered solution containing chelating agents.

[0020] In a further preferred embodiment, said buffered solutionconsists of 10 mM Tris-HCl (pH 8.0), and chelating agent is 1 mM EDTA.

[0021] In another preferred embodiment, the mechanical forces used areproduced by a device using horizontal orbital motion with or without arecessed platform (e.g. model Genie2 vortex from Fisher Scientific,Nepean, Ont., Canada), set to level 7, for 5 min.

[0022] In the above embodiments, there is no need for large particles(of the millimeter diameter size order) not involved in the lysis butinvolved in the motion of the lysing particles, such as those describedin WO 99/15621, WO 00/055338 and WO 02/10333. Instead of thesenon-lysing larger size particles, the present inventors have used amixture comprising granulometric gradients of small particles and largeparticles of comparatively bigger sizes (≈150-212 μm and 710-1180 μm).That mixture allows the user to be capable of performing a lysis stepper se within 5 minutes and without recourse to any mechanical aid suchas non-lysing big particles (larger than about 2 mm diameter size).

[0023] In a further preferred embodiment, the mechanical forces areproduced by a device using vertical angular motion (e.g. FastPrep®FP-120 apparatus from Qbiogene, Carlsbad, Calif., USA), set at powerlevel 6 for 45 seconds.

[0024] In another embodiment, said forces used to disrupt cells in thepresence of solid particles are transmitted by ultrasounds.

[0025] In order to control the inhibitors of NAT assays, one of thepreferred embodiments is heating the mix at temperatures ranging fromabout 55 to 100° C. for a sufficient time.

[0026] In another preferred embodiment, the lysis medium would alsocomprise a material binding or adsorbing NAT assays inhibitors, such asactivated carbon (charcoal, activated charcoal). This component would beadded or would be part of the bead mixture and would comprise differentparticle sizes, coarse synthetic beads coated with carbon, or aggregatedporous carbon beads. Those substances allow to adsorb porphyrin andporphyrin-like pigments or derivatives, hemin and hemin-like molecules,steroids and steroid-like hormones which are all known to be inhibitorsof NAT assays.

[0027] The NAT assays inhibitors may also be controlled simply bydiluting the sample, or by removal on density gradients, such asPercoll, or on adsorbent media (charcoal, etc.), or by freezing thesample. Combination of means or steps for controlling NAT assaysinhibitors are also within the scope of the present invention (forexample, heat+gradient, or heat+dilution, etc.)

DETAILED DESCRIPTION OF THE INVENTION

[0028] The Rapid Universal Cell Lysis and Nucleic Acids Preparation(RUCLANAP) method described in this invention forms the core ofprotocols used to prepare nucleic acids suitable for moleculardiagnostic applications based on nucleic acid detection. Modificationsmay be added depending on the nature of the test sample processed (seeExamples below), demonstrating the versatility of the method.

[0029] The sample of interest is first resuspended in a bufferedsolution which also includes a chelating agent. Tris(tris[hydroxymethyl]-aminomethane) is the preferred buffer here, sincein addition to its buffering power, Tris also contributes to theweakening of the lateral interaction between lipopolysaccharide (LPS)molecules in bacterial cell walls, by partially replacing other cationstightly bound to LPS. However, other buffers well known to biochemistscould be used instead. Even no buffer at all may be appropriate, as manysample types are naturally buffered; also, in some cases the assay maynot require a high sensitivity of detection. The chelating agent ispreferably EDTA (ethylenediaminetetraacetate), which chelates divalentcations, hence inhibiting nuclease enzymes by sequestering theiressential metallic cofactors; yet, other metal chelating agents couldalso be used instead. An additional effect of chelation is the loss ofcations involved in neutralization of the electrostatic repulsionbetween LPS molecules, resulting in the destabilization of the LPSmonolayer portion of the membrane. The observed increase in cellpermeability is likely to be due to the filling in of the space, formelyoccupied by LPS, by phospholipid molecules, thereby creatingphospholipid bilayer domains (Escherichia coli and Salmonella: Cellularand Molecular Biology, 1996, 2^(nd) Ed., Vol. I, Chapter 5, ASM Press,Washington).

[0030] The mixture is then put in contact with solid particles. Thepreferred volume of said mixture is 10 to 500 μL. Alternatively, saidparticles may already be present in the solution containing thechelating agent. Preferred solid particles consist of sterile,acid-washed glass beads placed into a 1.5-mL microtube. Solid particlescould be a mixture of particles having different sizes. The preferredmixture consists of two size ranges of beads, the first ranging from 150to 212 μm (70-100 U.S. sieve), and the second from 710 to 1180 μm (16-25U.S. sieve). Although in general small beads (150 to 425 μm) areefficient to lyse bacterial cells, efficient lysis of yeast cellsrequires larger beads (710 μm and up). Therefore, beside separate kits,compositions and methods tailored for one particular type ofmicroorganism, we devised a universal lysis mix, in which the two sizeranges of beads are mixed in a 4:1 ratio (w/w) and the total weight ofbeads is 0.05 g per microtube. This mix can efficiently lyse yeast cells(see Example 2). Alternatively, in the yeast lysis mix, the two sizeranges of beads are mixed in a 1:1 ratio (w/w) for a total weight of0.02 g of beads per microtube. Other particles found to be suitable forcell lysis include (but are not limited to) Ottawa sand and zirconiabeads.

[0031] Without being bound to any theory, we believe that using agranulometric gradient of particles having diameter sizes ranging from75 to about 2000 μm (namely 75 to 1500, even 75 to 1200) permits betterlysis. This is attributable to the presence of particles of variousdifferent sizes (the gradient) which increase the potential collisionalsurfaces to favor cell disruption and reduce the dead volume, hencefacilitating the recovery of the lysate. For the universal lysis ofdifferent microorganisms, having various sizes and shapes as bacteriaand yeast have, we discovered that combining two non-contiguousgranulometric gradients (150 to 212 μm and 710 to 1180 μm) wasparticularly efficient and convenient (see Example 1). Mixing whatothers called large non-lysing particles (WO 99/15621, WO 00/055338 andWO 02/10333) with a gradient of smaller lysing particles may also beused with the present invention.

[0032] Next, the viral, bacterial, fungal, parasitical, animal or humannucleic acids are released upon cell disruption by mechanical action.The latter could be provided by applying horizontal orbital motion(vortexing) on the microtube using a model Genie2 vortex from FisherScientific, for 1 to 5 min at power level 7. An alternate way to breakcells is to use the FastPrep® FP120 apparatus from Qbiogene, set atpower level 6 for 45 seconds. Other devices, including ultrasoundgenerators, may be suitable to apply sufficient mechanical energy tobreak cells.

[0033] The final step involves the control or neutralization ofpotential inhibitors of NAT assays, by heating the preparation. Thetemperature used may vary from 55 to 100° C., but preferably, theprotocol uses 95° C. The heating time may vary from a few seconds to 15min, depending on the sample, the usual duration being 2 to 5 min.Although heat is not absolutely required for some types of samples, auniversal protocol would preferably include this step for maximumperformance of the nucleic acids extraction.

[0034] Depending upon the nature of the sample, purification orconcentration or dilution steps can be added either before or afterlysis of the cells. Said steps may include, but are not limited to:buoyant density separation (with Percoll for instance, as described inExamples 4, 9 and 10), filtration, ultrafiltration, centrifugationand/or washing, ultracentrifugation, solid phase adsorption on carbon orother adsorbent material, chromatography, capture on magnetic particles,freezing, etc. All these steps intend to control the inhibitors of NATassays frequently present in biological samples. Freezing of theRUCLANAP-treated specimen has been shown to improve sensitivity in somecases; freezing is known to circumvent inhibition of nucleic acidamplification (Toye et al., 1998, J. Clin. Microbiol., 36:2356-2358;Templeton et al., 2001, Int. J. of STD & AIDS, 12:793-796).

[0035] The duration of agitation, and the ratios between the volume ofthe beads, the total volume of the sample and the total volume of therecipient are all parameters that may be adapted depending on the chosenagitation technique (magnetic, mechanical, sonicating). Examples ofstrategies to optimize lysis with particles may be found in WO 99/15621,WO 00/05338 and WO 02/10333. Optimization may also be performed for agiven type of recipient and agitation technique, by monitoring theefficiency of lysis and the sensitivity of the NAT assay conductedafterwards.

[0036] Unless specified otherwise, the contents of the cited referencesare incorporated to the present text by simply referring thereto.

LIST OF EXAMPLES

[0037] EXAMPLE 1: Core protocol for the RUCLANAP.

[0038] EXAMPLE 2: Comparison of RUCLANAP with eight commercial kits forrapid DNA extraction from microbial cultures.

[0039] EXAMPLE 3: Use of RUCLANAP for detection of Shiga toxin-producingbacteria.

[0040] EXAMPLE 4: Preparation of platelet concentrates for PCRamplification using RUCLANAP.

[0041] EXAMPLE 5: Use of RUCLANAP for the detection of group Bstreptococci in anal, vaginal, and combined vaginal/anal specimens.

[0042] EXAMPLE 6: Application of RUCLANAP for direct detection of S.saprophyticus from urine samples.

[0043] EXAMPLE 7: Detection of Candida albicans, Escherichia coli andStaphylococcus aureus in blood samples.

[0044] EXAMPLE 8: RUCLANAP for extraction of human DNA from clinicalsamples.

[0045] EXAMPLE 9: Effect of RUCLANAP on Bacillus subtilis endospores.

[0046] EXAMPLE 10: Detection of bacteria in bovine milk using RUCLANAP.

[0047] EXAMPLE 11: Use of RUCLANAP for the detection of Staphylococcusaureus in nasal swabs.

[0048] EXAMPLE 12: Direct detection of vancomycin-resistant enterococcifrom fecal samples using RUCLANAP.

[0049] EXAMPLE 13: Effect of variations in the ratios of glass beadssizes, or in total weight of beads, on RUCLANAP.

[0050] EXAMPLE 14: The effect of the particle size on lysis efficiency.

[0051] EXAMPLE 15: Effect on PCR of potentially inhibitory substancespresent in anal/vaginal samples.

[0052] EXAMPLE 16: Effect of bacterial density on RUCLANAP.

[0053] EXAMPLE 17: Total RNA extraction from Escherichia coli usingRUCLANAP.

[0054] EXAMPLE 18: Extraction of genomic DNA from Cryptosporidium parvumusing RUCLANAP.

[0055] EXAMPLE 19: Extraction of genomic DNA from Mycobacteriumsmegmatis using RUCLANAP.

[0056] EXAMPLE 20: RNA extraction from HIV-1 using RUCLANAP.

[0057] EXAMPLE 21: Use of zirconia beads for RUCLANAP.

EXAMPLES Example 1

[0058] Core protocol for the RUCLANAP. The sample (e.g. microbial cellsrecovered from an overnight broth culture after centrifugation, ormicrobial colonies grown on agar media, or microbial cells recoveredfrom a clinical sample) is resuspended in a buffered solution (10 to 50mM Tris-HCl, pH 8.0) which also includes a chelating agent (1 to 25 mMEDTA). The mixture (usually 10-100 μL) is then transferred to a 1.5-mL,screw-capped conical microtube (VWR Scientific Products, Mississauga,Ont., Canada) containing sterile, acid-washed glass beads. There are twosizes of beads, the first ranging from 150 to 212 μm (cat. no. G1145,Sigma, St-Louis, Mo., USA), and the second from 710 to 1180 μm (cat. no.G1152, Sigma). In the universal lysis mix, the two types of beads aremixed in a 4:1 ratio (w/w) and the total weight of beads is 0.05 g permicrotube; whereas in the yeast lysis mix, the beads are mixed in a 1:1ratio (w/w) for a total weight of beads of 0.02 g per microtube.

[0059] Next, the viral, bacterial, fungal, parasitical, animal or humannucleic acids are released when cells are disrupted by mechanicalaction. The latter is provided by vortexing the microtube on a modelGenie2 vortex from Fisher Scientific, for 1 to 5 min at power level 7.An alternate way to break cells is to use the FastPrep® FP120 apparatusfrom Qbiogene set at power level 6 for 45 seconds.

[0060] The final step involves the neutralization of potentialinhibitors of NAT assays by heating the preparation. The temperatureused may vary from 55 to 100° C., but preferably, the protocol uses 95°C. The heating time may vary from 1 to 15 min, the usual duration being2 to 5 min.

[0061] Using this protocol, we were able to extract nucleic acidssuitable for PCR amplification from a variety of microorganisms,including: Bacillus cereus, B. subtilis (vegetative cells andendospores), B. anthracis (vegetative cells, endospores and germinatingspores), Candida albicans, Candida dubliniensis, Candida krusei, Candidaparapsilosis, Candida tropicalis Clostridium difficile, Corynebacteriumaccolens, Corynebacterium genitalium, Corynebacterium jeikeium,Corynebacterium kutscheri, Corynebacterium minutissimum, Corynebacteriumstriatum, Crytosporidium parvum, Enterobacter cloacae, Enterococcusfaecalis, Enterococcus faecium, Escherichia coli, human immunodeficiencyvirus 1 (HIV-1), Klebsiella oxytoca, Klebsiella pneumoniae,Methanobrevibacter smithii, Micrococcus luteus, Mycobacterium smegmatis,Porphyromonas gingivalis, Porphyromonas gulae, Pseudomonas aeruginosa,Salmonella choleraesuis, Schizosaccharomyces pombe, Serratia marcescens,Shigella dysenteriae, Staphylococcus aureus, Staphylococcus epidermidis,Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcusmitis, Streptococcus mutans, Streptococcus parauberis, Streptococcuspneumoniae, Streptococcus pyogenes, Streptococcus salivarius,Streptococcus sanguinis, and Yersinia enterocolitica.

[0062] Nucleic acids extracted using RUCLANAP remain stable for severalmonths when stored frozen at −80° C. or −20° C., and may befreeze-thawed repeatedly (see Examples 3, 5, 11 and 12). Depending onthe starting sample, DNA preparations from clinical samples can be usedfor PCR amplification in the next 24 h when stored at 4° C. (see Example5), while DNA preparations from microbial cultures, stored at 4° C., canbe used for up to one week. In all the above storage conditions, nosignificant loss in PCR sensitivity has been observed.

Example 2

[0063] Comparison of RUCLANAP with eight commercial kits for rapid DNAextraction from microbial cultures. Eight commercial kits for rapid DNAisolation from microbial cultures were compared with the methoddescribed in the present invention. We have verified the efficiency ofthese kits to prepare DNA from the following microorganisms: (i) E. coliATCC 25922 (gram-negative bacterium), (ii) E. faecium ATCC 19434 and S.aureus ATCC 25923 (gram-positive bacteria) and (iii) C. albicans ATCC56884 (fungus). Broth cultures in mid-log phase of growth (OD₆₀₀ ofaround 0.5 for bacteria and 0.8 for fungi) were used as test samples.

[0064] Nine methods were thus evaluated by comparing their capacity toextract DNA suitable for PCR from the four different microorganisms. Thefollowing DNA extraction methods were executed according tomanufacturers instructions:

[0065] K1: Fast RNA™ blue Kit from Qbiogene;

[0066] K2: Dynabeads® DNA DIRECT™ from Dynal Biotech (Lake Success,N.Y., USA);

[0067] K3: NucliSens™ from Organon Teknika (bioMérieux, Marcy l'Étoile,France);

[0068] K4: GeneReleaser™ from BioVentures Inc (Murfreesboro, Tenn.,USA);

[0069] K5: High Pure™ PCR Template preparation Kit from Roche (Basel,Switzerland);

[0070] K6: Instagene™ Matrix from Bio-Rad Laboratories (Mississauga,Ont., Canada);

[0071] K7: QIAamp® Tissue Kit from Qiagen (Mississauga, Ont., Canada);

[0072] K8: Fast DNA™ Kit from Qbiogene;

[0073] K9: RUCLANAP.

[0074] The RUCLANAP protocol used for this experiment is described inExample 1, using the following conditions: chelating buffer=5×TE (50 mMTris-HCL, pH 8.0, 5 mM EDTA); glass beads=universal lysis mix;mechanical action=vortex at speel level 7 for 5 minutes. Few minormodifications of the original kit protocols were made in order tofacilitate comparison between the different methods. These modificationswere: (i) a volume of 100 μL of bacterial culture in mid-log phase wasalways used with each method, and (ii) the final volume of the DNApreparation was always adjusted to 100 μL. DNA extracts yielded witheach method were serially diluted to reach equivalents of 105 to 1genome copy/μL.

[0075] Conserved regions of the bacterial 16S rRNA or fungal , 18S rRNAgenes were used as targets for the amplification of DNA from bacteria orC. albicans. The 16S rRNA primers were described in our previous patent(SEQ ID NOs. 126 and 127, U.S. Pat. No. 5,994,066), whereas the 18S rRNAprimers were described in the literature (van Burik et al., 1998, J.Clin. Microbiol. 36: 1169-1175). Amplification reactions were performedin a 20 μL reaction mixture containing 50 mM KCl, 10 mM Tris-HCl (pH9.0), 0.1% Triton X-100, 2.5 mM MgCl₂, 0.4 μM each of the two selectedPCR primers, 200 μM each of the four deoxynucleoside triphosphates, 0.5U Taq DNA polymerase (Promega, Madison, Wis., USA) combined withTaqStart™ antibody (Clontech Laboratories Inc., Palo Alto, Calif., USA).Elimination of contaminating nucleic acids potentially present in thereagent mixture was carried out as described in the literature (Meier etal., 1993, J. Clin. Microbiol. 31: 646-652). One μL of diluted nucleicacid preparation was transferred into 19 μL of reaction mixture and wassubjected to thermal cycling (3 min at 94° C., and then 30 cycles of 30sec at 95° C. for the denaturation step, 30 sec at 55° C. for theannealing step, and 30 sec at 72° C. for the extension step, followed bya final extension step of 2 min at 72° C.) using a DNA engine PTC-200™thermal cycler (MJ Research Inc., Waltham, Mass., USA).

[0076] Ten μL of the PCR-amplified reaction mixture was analyzed byelectrophoresis at 170 V for 30 min, in a 2% agarose gel containing 0.5μg of ethidium bromide per mL. The size of the amplification productswas estimated by comparison with a 50-bp molecular size standard ladder.

[0077] As shown in Table 1, the highest efficiency for DNA recovery wasobtained with K9 (the RUCLANAP method described in the presentinvention), followed in order by K1, K5, K7, K6, K3, K8, K2, and K4. Thedifference in efficiency between kits ranged from 0.5 to 4 log,depending on the microbial species tested. When analyzed on the basis ofmanipulation steps and time requirement, the most convenient kit was K9,requiring 15 min in a 5 step protocol, followed by K2, K6, K1, K8, K4,K5, K3, and K7. These extraction methods required 15 to 150 min forcompletion, in 4 to 40 steps protocols. Overall, the most efficient andsimplest method for rapid DNA extraction from microbes was RUCLANAP.

Example 3

[0078] Use of RUCLANAP for detection of Shiga toxin-producing bacteria.This assay was published in the Journal of Clinical Microbiology, 2002,4:1436-1440. No information regarding the sample preparation protocolwas disclosed in this publication. Shiga toxin-producing Escherichiacoli and Shigella dysenteriae cause bloody diarrhea andhaemolytic-uremic syndrome. Currently, identification relies mainly onthe phenotypic identification of S. dysenteriae and E. coli serotypeO157:H7. However, other serotypes of E. coli are increasingly found tobe producers of type 1 and/or type 2 Shiga toxins. Two pairs of PCRprimers targeting highly conserved regions present in each of the Shigatoxin genes stx1 and stx2 were designed to amplify all variants of thosegenes (described in our previous patent publication WO 01/23604). Thefirst primer pair, oligonucleotides SEQ ID NO. 1080 and 1081, yields anamplification product of 186 bp from the stx1 gene. For this amplicon,the molecular beacon SEQ ID NO. 1084 (patent publication WO 01/23604)was designed for the specific detection of stx1 using the fluorescentlabel 6-carboxy-fluorescein. A second pair of PCR primers,oligonucleotides SEQ ID NO. 1078 and 1079, yields an amplificationproduct of 160 bp from the stx2 gene. Molecular beacon SEQ ID NO. 1085(patent publication WO 01/23604) was designed for the specific detectionof stx2 using the fluorescent label 5-tetrachloro-fluorescein. Bothprimer pairs were combined in a multiplex PCR assay.

[0079] Stool samples were prepared in the following manner. For solidfecal material, a sterile swab was dipped into the sample, thentransferred to a 1.5-mL screw-capped microtube containing 1 mL of TEbuffer (10 mM Tris-HCl, 1 mM EDTA). The stem of the swab was wrapped ingauze and bent until it broke, so that the microtube, containing theinoculated portion of the swab, could be closed. Fecal material wasresuspended in solution by vortexing vigourously at least 15 seconds. A50 μL aliquot was transferred to a 1.5-mL, screw-capped microtubecontaining 0.05 g of sterile, acid-washed glass beads (universal lysismix, as described in Example 1). For liquid fecal material, a 50 μLaliquot of liquid stools was transferred directly to the microtubecontaining beads. Subsequently, the microtube was vortexed for 5 min atspeed level 7 on a Genie2 model vortex from Fisher Scientific. After aquick centrifuge spin, the microtube was heated for 5 min at 95° C. A 2min. centrifugation at 10 000 g followed. The supernatant wastransferred to another microtube, and 1.5 μL of 1:10 and 1:20 dilutionswere used in PCR reactions. Extracts could be stored for at least oneweek at −20° C. without any significant loss in PCR analyticalsensitivity.

[0080] PCR amplification was carried out using 0.8 μM of each primer SEQID NO. 1080 and 1081, 0.5 μM of each primer SEQ ID NO. 1078 and 1079,0.3 μM of each molecular beacon, 8 mM MgCl₂, 490 μg/mL bovine serumalbumin (BSA), 0.2 mM dNTPs (Amersham Biosciences, Uppsala, Sweden), 50mM Tris-HCl, 16 mM NH₄SO₄, 1×TaqMaster (Eppendorf, Hamburg, Germany),2.5 U KlenTaq1 DNA polymerase (AB Peptides, St. Louis, Mo., USA) coupledwith TaqStart™ antibody (Clontech), and 1.5 μL of the prepared stoolsample in a final reaction volume of 25 μL. PCR amplification wasperformed using a SmartCycler® thermal cycler (Cepheid, Sunnyvale,Calif., USA). The optimal cycling conditions for maximum sensitivity andspecificity were 60 seconds at 95° C. for initial denaturation, then 45cycles of three steps consisting of 10 seconds at 95° C., 15 seconds at56° C. and 5 seconds at 72° C. Detection of the PCR products was made inreal-time, by measuring the fluorescent signal emitted by the molecularbeacon when it hybridizes to its target at the end of each annealingstep at 56° C.

[0081] The assay was specific for the detection of both toxins, asdemonstrated by the perfect correlation between PCR results and thephenotypic characterization performed using antibodies specific for eachShiga toxin type. The assay was successfully performed on several Shigatoxin-producing strains isolated from various geographic areas of theworld, including 10 O157:H7 E. coli, 5 non-O157:H7 E. coli and 4 S.dysenteriae. The detection limit for this PCR assay was around 10⁵colony forming units (CFU) per gram of fecal material. The assay wasvalidated by testing 38 fecal samples obtained from 27 patients. Ofthese samples, 26 were positive for stx1 and/or stx2. Compared with theculture results, both the sensitivity and the negative predictive valuewere 100%. The specificity was 92% and the positive predictive value was96%. RUCLANAP worked efficiently with both liquid or solid stools, andwhether or not they contained traces of blood.

Example 4

[0082] Preparation of Platelet Concentrates for PCR Amplification UsingRUCLANAP. Blood platelet preparations need to be monitored for bacterialcontaminations, as the latter may develop during storage. Primers weredesigned from tuf gene fragments. Oligonucleotide sequences conservedfor the 17 major bacterial contaminants of platelet concentrates werechosen (oligonucleotides SEQ ID NO. 636, 637, 553 and 575, described inour previous patent publication WO 01/23604, yielding amplificationproducts of 245 bp and 368 bp), thereby permitting the detection ofthese bacterial species. The 104 bacterial species whose genomic DNA isdetected efficiently with the assay are listed in Table 14 of ourprevious patent publication WO 01/23604.

[0083] Detection of these PCR products was made on the LightCycler™thermocycler (Roche) using SYBR® Green I (Molecular Probes Inc., Eugene,Oreg., USA), a fluorescent dye that binds to double-stranded DNA.

[0084] Platelet concentrates (250 μL), spiked with each of the 17 majorbacterial contaminants, were submitted to buoyant density separationover 600 μL of Percoll SIM-40 (40% Percoll (Amersham Biosciences) and60% peptone water (BD Microbiology Systems, Cockeysville, Md., USA)) ina 1.5 mL screw-capped microtube containing glass beads (universal lysismix, see Example 1). The mixture was centrifuged 75 seconds at 16 200 g.The supernatant was removed, and the pellet was mixed by inversion with1 mL of 0.01 M phosphate-buffered saline (PBS), pH 7.4. Aftercentrifugation at 10 000 g for 5 min, the supernatant was removed, andthe pellet was resuspended in 20 μL of TE or ultrapure water. Cells weredisrupted in the FastPrep® FP-120 apparatus (Qbiogene) for 45 seconds atpower level 6. After a quick spin, the microtube was heated 2 min at 95°C. One μL of each preparation was used for PCR amplification.

[0085] Fluorogenic detection of PCR products with the LightCycler™ wascarried out using 1.0 μM of each universal primer and 0.4 μm of eachStaphylococcus-specific primer, 4.5 mM MgCl₂, 0.4 mg/mL BSA, 0.2 mMdNTPs (Amersham Biosciences), PC2 buffer (50 mM Tris-HCl, 3.5 mM MgCl₂,BSA 0.15 mg/mL, 16 mM NH₄(SO₄)₂); 1.875 U KlenTaq DNA polymerase (ABPeptides) coupled with TaqStart™ antibody (Clontech), and 0.5×SYBR®Green I (Molecular Probes, Inc.); in a final volume of 15 μL.Elimination of contaminating nucleic acids potentially present in thereagent mixture was carried out as described above. The optimal cyclingconditions for maximum sensitivity and specificity were 1 minute at 94°C. for initial denaturation, then forty-five cycles of three stepsconsisting of 0 second at 95° C., 5 seconds at 60° C. and 9 seconds at72° C. Amplification was monitored during each elongation cycle bymeasuring the level of fluorescence, and after amplification, meltingcurve analysis was performed. With this assay, DNA from all prominentbacterial contaminants of platelet concentrates was extracted usingRUCLANAP. Analytical sensitivity tests were performed on 9 frequentbacterial contaminants of platelets: Enterobacter cloacae, Bacilluscereus, Salmonella choleraesuis, Serratia marcescens, Pseudomonasaeruginosa, Staphylococcus aureus, Staphylococcus epidermidis,Escherichia coli and Klebsiella pneumoniae. The detection limit was ≦20CFU/mL of platelet concentrates for all of these 9 species tested.

Example 5

[0086] Use of RUCLANAP for the detection of group B streptococci inanal, vaginal, and combined vaginal/anal specimens. The development ofconventional and real-time PCR assays for the rapid detection of group Bstreptococci was initially published in Clinical Chemistry, 2000, 46:324-331. Subsequently, a clinical study based on these assays has beendescribed in the New England Journal of Medicine, 2000, 343: 175-179. Noinformation regarding the sample preparation protocol was disclosed inthese publications. The efficacy of two polymerase-chain-reaction (PCR)assays for routine screening of pregnant women for group B streptococciat the time of delivery was studied. Anal, vaginal, and combinedvaginal/anal specimens were obtained from 112 pregnant women; for 57women, specimens were obtained before and after rupture of the amnioticmembranes. The specimens were tested for group B streptococci (i) byculture in a standard selective broth medium, (ii) with a conventionalPCR assay, and (iii) with a fluorogenic PCR assay.

[0087] The rapid lysis protocol used to prepare clinical samples for DNAamplification in these studies was the following:

[0088] A. The BBL Culturette™ swab (BD Microbiology Systems) used tosample anal, vaginal or vaginal-anal surfaces was inserted in a 1.5-mL,screw-capped microtube containing 300 μL GNS medium (Todd-Hewitt brothsupplemented with 8 μg/mL of gentamicin and 15 μg/mL of nalidixic acid).The tip of the swab was rolled around the tube to expel the maximum ofliquid.

[0089] B. A 10 μL aliquot was transferred to a 1.5-mL, screw-cappedmicrotube containing 0.05 g of sterile, acid-washed glass beads(universal lysis mix, as described in Example 1), to which 40 μL of TEbuffer were added. The microtube was vortexed for 5 min at speed level 7on a Genie2 model vortex from Fisher Scientific.

[0090] C. After a quick centrifuge spin, the microtube was heated for 2min at 95° C.

[0091] D. 1.5 μL of the mixture was used directly in the PCR reaction.Extracts could be stored for several months at −20° C. without anysignificant loss in PCR analytical sensitivity.

[0092] Based on culture, the combined vaginal/anal specimens from 33 ofthe 112 women (colonization rate of 29.5%) were positive for group Bstreptococci. The two PCR assays detected group B streptococcalcolonization in specimens from 32 of these 33 women: the one negativePCR result was a sample obtained after the rupture of membranes. Ascompared with the culture results, the sensitivity of both PCR assayswas 97.0% while the negative predictive value was 98.8%. Both thespecificity and the positive predictive value of the two PCR assays were100%. The length of time required to obtain results was 30 to 45 min forthe fluorogenic PCR assay, 100 min for the conventional PCR assay, andat least 36 hours for the culture method.

[0093] Alternatively, the above protocol was also used in the followingmanner:

[0094] A. The BBL Culturette™ swab (BD Microbiology Systems) used tosample anal, vaginal or vaginal-anal surfaces was inserted in a 1.5 mL,screw-capped microtube containing 1 mL of TE buffer. The stem of theswab was wrapped in gauze and bent until it broke, so that the microtubecontaining the inoculated portion of the swab could be closed.

[0095] B. Cells were resuspended in solution by vortexing vigorously atleast 15 seconds. A 50 μL aliquot was transferred to a 1 .5-mL,screw-capped microtube containing 0.05 g of sterile, acid-washed glassbeads (universal lysis mix, as described in Example 1). The microtubewas vortexed for 5 min at speed level 7 on a Genie2 model vortex fromFisher Scientific.

[0096] C. After a quick centrifuge spin, the microtube was heated for 2min at 95° C.

[0097] D. 1.5 μL of the mixture was used directly in the PCR reaction.

[0098] The efficiency of Streptococcus agalactiae lysis by the RUCLANAPmethod was estimated to 99.99%, as determined by viable counts.

Example 6

[0099] Application of RUCLANAP for direct detection of S. saprophyticusfrom urine samples. This assay was published in the Journal of ClinicalMicrobiology, 2000, 38: 3280-3284. No information regarding the samplepreparation protocol was disclosed in this publication. Staphylococcussaprophyticus is one of the most frequently encountered microorganismsassociated with acute urinary tract infections (UTIs) in young, sexuallyactive female outpatients. Conventional identification methods based onbiochemical characteristics can efficiently identify S. saprophyticus,but the rapidity of these methods needs to be improved. Rapid and directidentification of this bacterium from urine samples would be useful toaccelerate the diagnosis of S. saprophyticus infections in the clinicalmicrobiology laboratory. A PCR-based assay for the specific detection ofS. saprophyticus has been developed. An arbitrarily primed PCRamplification product of 380 bp specific for S. saprophyticus wassequenced and used to design a set of S. saprophyticus-specific PCRamplification primers (SEQ ID NO. 1208 and 1209 in patent publication WO01/23604). The PCR assay, which uses standard agarose gelelectrophoresis for amplicon analysis, was specific for S. saprophyticuswhen tested with DNA from 49 gram-positive and 31 gram-negativebacterial species. This assay was also able to amplify DNA efficientlyfrom all 60 strains of S. saprophyticus tested, which originated fromvarious geographical areas. This assay was adapted for direct detectionfrom urine samples.

[0100] The protocol used for the rapid lysis of bacterial cells for therelease of nucleic acids from urine samples was the following: 500 μL ofurine samples spiked with various amounts of S. saprophyticus cells weretransferred to a 1.5-mL, screwcapped microtube containing 0.05 g ofsterile, acid-washed glass beads (universal lysis mix, as described inExample 1). After centrifugation for 5 min at 15 800 g, the supernatantwas discarded and the pellet was washed with 1 mL of PBS. After anothercentrifugation for 5 min at 15 800 g, the supernatant was discarded.Then, 50 μL of TE or PBS were added to the tube, and the sample wasresuspended and lysed by vortexing for 5 min at speed level 7 on aGenie2 model vortex from Fisher Scientific. A heating step of 2 min at95° C. followed. One μL of prepared urine sample was added directly to aPCR reaction. The analytical sensitivity levels achieved withRUCLANAP-treated urine samples were 0.3-1.4 CFU/PCR reaction with 40cycles of amplification. By contrast, spiked urine samples addeddirectly to the PCR (without treatment) showed a detection limit of700-800 CFU/PCR. This PCR assay for the specific detection of S.saprophyticus is simple and rapid (approximately 90 min, including theapproximately 10 minutes required for urine specimen preparation).

Example 7

[0101] Detection of Candida albicans, Escherichia coli andStaphylococcus aureus in blood samples. We devised a procedure to detectfungal and bacterial nucleic acids from infected blood. As bloodcontains large amounts of potent PCR inhibitors, rapid washes arerequired prior to the RUCLANAP protocol. Blood samples (1 mL) spikedwith various amounts of bacterial or fungal cells were added to 1.5 mLmicrotubes containing glass beads for the universal or yeast lysis(Example 1). Three sequential washes were then performed by adding 1 mLof TE buffer to the tube and mixing briefly, with a centrifugation step(21 000 g for 1 min) and removal of the supernatant by aspirationbetween each wash. After the last wash, a quick centrifuge spin wasperformed and the residual liquid was removed by gentle aspiration. 12.5μL of 5×TE buffer were added to the beads, and cells were then disruptedin the FastPrep® FP-120 apparatus for 45 seconds at power level 6. Aftera quick centrifuge spin, the microtube was heated 2 min at 95° C. andthen centrifuged briefly. 2 to 4 μL of the preparation were used for PCRamplification. Using species-specific PCR assays described in ourprevious patent publication WO 01/23604, we obtained the followingdetection limits: 40 CFU/mL of blood for Escherichia coli, 30 CFU/mL ofblood for Staphylococcus aureus, and 50 CFU/mL of blood for Candidaalbicans. Omission of the final heating step resulted in a loss insensitivity, demonstrating that PCR inhibitors still present in washedblood samples can be successively eliminated by heat.

Example 8

[0102] RUCLANAP for extraction of human DNA from clinical samples.Although our goal is to detect DNA from microorganisms, human DNA isextracted as well from the clinical samples described in Examples 3 to7, 11, 12, 15 and 17. For example, using primers specific to theβ-globin gene (Swan et al., 1999, J. Clin. Microbiol. 37: 1030-1034), wecould detect from 1 to 104 copies of this human genetic target in rectalswabs treated with RUCLANAP, as described in Example 12.

[0103] RUCLANAP cannot only be useful for genetic purposes, but is alsouseful to monitor the efficiency of the sampling procedure. The validityof individual samples can be monitored by measuring the human DNA loadon the swab used for sampling, and comparing the result with a definedthreshold.

Example 9

[0104] Effect of RUCLANAP on Bacillus subtilis endospores. Recovery ofDNA from bacterial endospores has been described, but the tediousmethods used in many studies resulted in severely sheared DNA, whichhindered PCR sensitivity (Kuske et al., 1998, Appl. Environ. Microbiol.64: 2463-2472). These authors have demonstrated that bead millhomogenization was the only effective method to extract DNA frombacterial endospore preparations. We demonstrate here the efficacy ofRUCLANAP on spores.

[0105] The following protocol for endospore enrichment and purificationwas adapted from the method of Belgrader et al. (Analytical Chemistry1999, 71: 4232-4236). A 50-mL culture of Bacillus subtilis grown insporulation medium (Holt and Krieg, Enrichment and Isolation, In:Methods for General and Molecular Bacteriology, 1994, American Societyfor Microbiology, Washington, D.C.) was vortexed, then separated in twovolumes of 25 mL. After centrifugation at 2500 g for 20 min, the pelletswere washed three times with 5 mL of sterile distilled water, each washbeing centrifuged at 2500 g for 10 min. The final pellets wereresuspended in 4 mL of 50 mM NaCl, 100 mM EDTA, pH 6.9. Lysozyme wasadded at a final concentration of 1 mg/mL, and the mixture was incubatedat 30° C. for 90 min. After two additional washes as described above,the pellets were resuspended in 1 mL of 5% Triton X-100.

[0106] Next, 250 μL of the above endospore suspension were submitted tobuoyant density separation over 600 μL of Percoll SIM-40 (40% Percolland 60% peptone water) in a 1.5 mL screw-capped microtube. The mixturewas centrifuged 15 min at 750 g. The pellet was washed three times with250 μL of sterile water, each wash being centrifuged at 9300 g for 2min. The final pellet was resuspended in 100 μL of sterile distilledwater. The final purified spore suspension was serially diluted 1/10 toa final factor of 1×10⁻⁸. 100 μL of each dilution were either platedonto blood agar plates for bacterial count, or placed in a hemacytometerfor endospore count. The final purified spore suspension consisted of1.73×10¹⁰ endospores/mL, which corresponded to 5.7×10⁹ CFU/mL.

[0107] The RUCLANAP method was adapted for endospore lysis in thefollowing manner. A volume of 100 μL of each serial dilution of purifiedendospores was centrifuged 5 min at 9300 g, the pellet was resuspendedin 40 μL of TE buffer, and transferred to a 1.5 mL screw-capped tubecontaining glass beads (universal lysis mix, see Example 1). Endosporeswere disrupted in the FastPrep® FP-120 apparatus for 45 seconds at powerlevel 6. After a quick centrifuge spin, the microtube was heated for 5min at 95° C. Subsequently, 100 μL of a 1/10 dilution of the lysedmaterial was plated onto blood agar plates to assay endospore lysis. Theefficiency of lysis of Bacillus subtilis endospores by RUCLANAP wasestimated at 98.8% based on CFU counts performed before and after thelysis treatment.

[0108] One μL of a 1/10 dilution of the lysed material was used for PCRamplification. The PCR assay used for universal bacterial detectionusing primers SEQ ID NO. 643 and 644 has been described in our previouspatent publication WO 01/23604. The detection limit determined withuntreated endospores was in the range of 1.3×10⁵ to 1.7×10⁶endospores/mL, whereas the detection limit of endospores lysed withRUCLANAP was in the range of 1.3×10³ to 1.7×10⁴ endospores/mL(approximately 100 times better). In each PCR reaction tube, we coulddetect 3 to 43 RUCLANAP-treated endospores, compared to a detectionlimit of 320 to 4340 for untreated endospores. Further concentration ofspore suspensions before RUCLANAP can allow to lower the detection limitin terms of CFU per mL.

Example 10

[0109] Detection of bacteria in bovine milk using RUCLANAP. Clinicalmastitis is a common disease in dairy cows in Canada; approximately 1 in5 lactating cows has at least one episode of clinical mastitis in itslifetime. As costs for each case are estimated to 150-200 $CAN, a crudeestimate of losses to the Canadian dairy industry amounts to 44 million$CAN annually; in the U.S., the estimation is about $1.8 billion. Themost common bacterial mastitis pathogens are Staphylococcus sp.,Streptococcus sp., and coliforms (Sargeant et al., 1998, Can. Vet J. 39:33-38). We have designed a protocol based on the RUCLANAP method todetect bacteria in bovine milk.

[0110] The milk samples were first incubated at 37° C. for 6 hours. Theprotocol, using Percoll™ buoyant density separation, to extract nucleicacids from bovine milk was as described in Example 4, using a totalweight 0.01 g of glass beads solely composed of the 150-212 μm size. Arange of 80 to 100% of the milk samples positive by culture forStaphylococcus aureus or Streptococcus agalactiae were detected by ourspecies-specific PCR assays targetting these two bacterial species(assays described in Examples 11 and 5, respectively).

Example 11

[0111] Use of RUCLANAP for the detection of Staphylococcus aureus innasal swabs. One of the PCR assays described by Martineau et al. (2000,J. Antimicrob. Chemother. 46: 527-534) has been specifically used todetect Staphylococcus aureus from nasal swabs, using RUCLANAP to extractthe nucleic acids. The protocol used was the following:

[0112] A. The BBL Culturette™ swab (BD Microbiology Systems) used tosample nasal mucosa was inserted into a 1.5-mL, screw-capped microtubecontaining 300 μL of TE buffer, supplemented with 1.5 mg/mL of BSA. Thestem of the swab was wrapped in gauze and bent until it broke, so thatthe microtube containing the inoculated portion of the swab could beclosed.

[0113] B. Cells were resuspended in solution by vortexing 1 min. A 50 μLaliquot was transferred to a 1.5-mL, screw-capped microtube containing0.05 g of sterile, acid-washed glass beads (universal lysis mix, asdescribed in Example 1). The microtube was vortexed for 5 min at speedlevel 7 on a Genie2 model vortex from Fisher Scientific.

[0114] C. After a quick centrifuge spin, the microtube was heated for 2min at 95° C.

[0115] D. After a quick centrifuge spin, 2 μL of the mixture was useddirectly in the PCR reaction using the Staphylococcus aureus-specificPCR assay originally described by Martineau et al. (1998, J. Clin.Microbiol. 36:618-623). Extracts could be stored for several months at−80° C. without any significant loss in PCR sensitivity.

[0116] In a preliminary study with this PCR assay, 81 nasal swabs wereprocessed using the RUCLANAP protocol. A total of 36 samples wereculture-positive for Staphylococcus aureus, and 35 of those samples weredetected by our PCR assay, for a sensitivity of 97.2%. The heating stepwas essential to remove PCR inhibitors present in the nasal samples.

Example 12

[0117] Direct detection of vancomycin-resistant enterococci from fecalsamples using RUCLANAP. The increasing incidence of nosocomialvancomycin-resistant enterococci (VRE) outbreaks attests to theimportance of early identification of VRE-colonized patients to preventperson-to-person transmission. Routine culture methods for the detectionof VRE from stool or rectal samples are reliable, but require at least 2to 4 days for completion. Therefore, we investigated the use of aPCR-based assay coupled with capture-probe hybridization as analternative for rapid detection of VRE directly from fecal samples. Thelatter were collected during a VRE hospital outbreak in Quebec City inJanuary 2000.

[0118] Fecal material from 533 stools or rectal swabs were analyzed byi) standard culture method using selective agar media and ii) our PCRassay coupled with capture-probe hybridization. The PCR assay wasperformed directly from the fecal material prepared with the RUCLANAPmethod, in a manner similar to that described in Example 3. For stools,a sterile swab was dipped into the sample, then transferred to a 1.5-mLscrew-capped microtube containing 1 mL of TE buffer. The stem of theswab was wrapped in gauze and bent until it broke, so that the microtubecontaining the inoculated portion of the swab could be closed. Rectalswabs were directly transferred to a 1.5-mL screw-capped microtube inthe same manner. Fecal material was resuspended in solution by vortexingvigourously for 1 min. A 50 μL aliquot was transferred to a 1.5-mLscrew-capped microtube containing 0.05 g of sterile, acid-washed glassbeads (universal lysis mix, as described in Example 1). Subsequently,the microtube was vortexed for 5 min at speed level 7 on a Genie2 modelvortex from Fisher Scientific. After a quick centrifuge spin, themicrotube was heated for 2 min at 95° C. Then, 2 μL of 1:2.5, 1:5 and1:10 dilutions were used in PCR reactions. Extracts could be stored forseveral months at −80° C. without any significant loss in PCRsensitivity.

[0119] A multiplex PCR assay consisting of vanA- and vanB-specificprimer pairs was developed and coupled with post-PCR hybridization usingtwo capture probes targeting the respective vanA and vanB amplicons.Primers and probes used in this multiplex PCR assay are described inExample 23 of our previous patent WO 01/23604.

[0120] Twenty-nine specimens were positive for VRE (all Enterococcusfaecium) based on culture. PCR detected 28 of the 29 culture-positivespecimens of which i) 26 were positive for vanA, and ii) 2 were positivefor both vanA and vanB. Among the culture-negative specimens, PCRdetected one additional vanA-positive and 10 vanB-positive specimens.Overall, the multiplex PCR assay had a sensitivity of 96.6% and aspecificity of 97.9%. This assay based on RUCLANAP extraction representsa promising and rapid screening test for the detection of VRE from fecalsamples.

Example 13

[0121] Effect of variations in the ratios of glass beads sizes, or intotal weight of beads, on RUCLANAP. In order to verify if modificationsin a) the ratio of glass beads sizes or b) total weight of beads used inthe universal lysis mix had an effect on the sensitivity of the PCRdetection, the following experiments were designed and performed. First,different bead ratios were compared. In the universal lysis mix, theratio of small beads (150-212 μm) to large beads (710-1180 μm) is 4:1,and there are 40 mg of small beads+10 mg of large beads per 1.5 mLreaction tube. Two other bead ratios were tested for comparison whilekeeping the total weight of beads to 50 mg:

[0122] 3.5:1.5 (35 mg of small beads+15 mg of large beads)

[0123] 4:1 (40 mg of small beads+10 mg of large beads, universal mix)

[0124] 4.5:0.5 (45 mg of small beads+5 mg of large beads).

[0125] Next, different combinations of small/large beads ratios andtotal beads weight were compared:

[0126] 1:1 (10 mg of small beads+10 mg of large beads, total 20 mg)

[0127] 2.5:1 (25 mg of small beads+10 mg of large beads, total 35 mg)

[0128] 4:1 (40 mg of small beads+10 mg of large beads, total 50 mg,universal mix)

[0129] 5.5:1 (55 mg of small beads+10 mg of large beads, total 65 mg).

[0130] Finally, different total weights of small beads only were tested:20 mg, 35 mg, 50 mg, and 65 mg.

[0131] A volume of 50 μL of serial tenfold dilutions of a mid-logculture of Streptococcus agalactiae ATCC 13813 was added to thedifferent mixes of beads. The microtube was vortexed for 5 min at speedlevel 7 on a Genie2 model vortex from Fisher Scientific. After a quickcentrifuge spin, the microtube was heated for 2 min at 95° C. Then, 1.5μL of the mixture was used directly in the PCR reaction, which wasdescribed in Example 5.

[0132] No significant difference in the analytical sensitivity wasobserved for all the above bead ratios and weights tested.

Example 14

[0133] The effect of the particle size on lysis efficiency. The effectof the particle size on lysis efficiency was also tested on Candidaalbicans, Escherichia coli, Enterococcus faecalis, Staphylococcusaureus, and Micrococus luteus.

[0134] Bacteria were grown on blood agar at 37° C. overnight. Yeastswere grown on Sabouraud at 30° C. overnight. Cells were resuspended inPBS in order to obtain 10⁸ cells per ml (for yeasts, a reading of 0.23on a Dade Behring nephelometer (Deerfield, Ill., USA), and for bacteria,a reading of 0.08).

[0135] In order to evaluate the effect of particle size on theefficiency of lysis of microbial cells, various types and sizes of solidparticules were tested: 0.2 g of glass beads from Sigma (<106 μm,150-212 μm, 212-300 μm, 425-600 μm, or 710-1180 μm), 0.2 g of a mixtureof all of the preceding glass bead sizes, or 0.2 g of Ottawa sand.

[0136] For yeasts, the best analytical sensitivity was obtained whenusing the biggest particles (710-1180 μm) as well as with the mixture ofbeads of all sizes. The next best results were obtained with sand,followed by beads of medium size.

[0137] For bacteria, small size beads of 150-212 μm as well as themixture of all bead sizes yielded the best results. Again, sand was lessefficient.

[0138] The results obtained suggest that for efficient microbial celllysis, the use of beads of all sizes, or of at least two sizes selectedwithin the ranges of about 710-1180 μm and about 150-212 μm, isrequired. These sizes (the whole range thereof or the two more specificones) would allow a better reproducibility of the lysis method overOttawa sand, because the latter may vary from lot to lot, ultimatelyaffecting the efficiency of cell lysis and consequently the analyticalsensitivity of the NAT assay.

Example 15

[0139] Effect on PCR of potentially inhibitory substances present inanal/vaginal samples. The use of RUCLANAP for the detection of group Bstreptococci (GBS) was described in Example 5. A number of substanceshaving PCR inhibitory potential may be found in anal, vaginal orcombined vaginal/anal samples upon sampling in pregnant women justbefore labor. Those substances include urine, amniotic fluid, bloodoriginating from the umbilical cord, vaginal secretions, meconium,stools and lubricant (K-Y® lubricating jelly from Johnson & Johnson,Markham, Ont., Canada). In order to assess their inhibitory potential,swabs were dipped in several samples of each of these pure substances,and the RUCLANAP protocol described at the end of Example 5 wasperformed. The GBS real-time PCR assay was then performed with 1.5 μL ofprocessed sample (pure or diluted 1:1 in ultrapure water) in thepresence of 100 copies of the internal control template (Ke et al.,2000, Clin. Chem. 46: 324-331). Inhibition was assessed by comparing thefluorescence emitted by the internal control in the presence of each ofthoses inhibitory substances relative to a negative control (noinhibitory sample added). For all substances tested, the real-time PCRfluorescent signal was equivalent to the signal of the negative control,showing that the RUCLANAP procedure is able to control efficientlyvarious PCR inhibitory substances.

Example 16

[0140] Effect of bacterial density on RUCLANAP. To determine if thebacterial density may affect the efficacy of RUCLANAP, two protocolswere tested. In the first protocol, serial dilutions were performed onthe bacterial sample before lysis with RUCLANAP, while in the secondprotocol, the bacterial sample was undiluted but dilutions wereperformed on the lysed material. The starting sample was obtained bypreparing a bacterial suspension in PBS, calibrated to obtain 10⁸bacteria per mL, using Staphylococcus aureus colonies grown overnight onan agar plate.

[0141] Serial 1/10 dilutions of the suspension were made in PBS, untilobtaining 10³ bacteria/mL. A volume of 100 μl of the initial suspensionor of each ten-fold dilution was mixed with beads of sizes150-212+710-1180 μm (universal lysis mix, see Example 1). The mixturewas placed in the FastPrep® FP-120 apparatus, and shaken at speed 6 for45 seconds. Lysed material from the initial suspension (10⁸ bacteria permL) was serially diluted (ten-fold dilutions) in TE buffer until a finaldilution factor of 1/100 000 was obtained. A PCR was conducted withuniversal primers (as described in Example 12 from our previous patentWO 01/23604), using 1 μL of lysed sample (or diluted lysed sample).

[0142] There was no significant difference in the sensitivity betweenthe two protocols, indicating that at least in the range tested here,the density of bacterial population in the test sample does not affectsignificantly lysis efficacy.

Example 17

[0143] Total RNA extraction from Escherichia coli using RUCLANAP. Thisexperiment was designed to demonstrate that RNA, suitable for cDNAsynthesis and amplification by RT-PCR, may be extracted from bacterialcells using RUCLANAP. RUCLANAP was adapted for RT-PCR by adding RNaseinhibitor. A volume of 50 μL of broth culture of E. coli ATCC 25922 inmid-log phase of growth (O.D.₆₀₀=0.5) was centrifuged in a 1.5 mLmicrotube containing the universal mix of beads, and the cell pellet wasresuspended in 50 μL of 5×TE buffer containing 40 U of recombinantribonuclease inhibitor (RNasin®, Promega). The basic RUCLANAP protocolwas then followed (Example 1), using the universal lysis mix. Serialtenfold dilutions of the lysate were performed in TE buffer.

[0144] Next, 10 μL of diluted lysate was mixed with 10 μL of digestionbuffer (20 mM Tris-HCl pH 8.0, 5 mM MgCl₂, 0.2 mM CaCl₂) and incubatedfor 1 min at 37° C., with either 10 U of DNase I (RNase-free, Roche), or0.14 U of RNase A (DNase-free, Qiagen), or both enzymes DNase I andRNase A, or no enzyme at all. An enzyme inactivation step of 5 min at95° C. followed for all conditions tested. One μL of treated lysate wasthen added to the RT-PCR or the PCR reaction.

[0145] The Superscript™ One-Step RT-PCR with Platinum® Taq fromInvitrogen (Carlsbad, Calif., USA) was used according to themanufacturer's instructions, in a final volume of 50 μL. Both steps,reverse transcription and PCR, were performed successively in the samereaction tube. Primers targeting the tuf gene and specific to theEscherichia genus were used (SEQ ID. NO. 1661 and 1665 in patent WO01/23604). cDNA synthesis was accomplished by a 30 min incubation at 50°C., followed by an initial denaturation step of 2 min at 94° C. For thesubsequent PCR amplification, 40 cycles of 15 s at 94° C., 30 s at 56°C. and 30 s at 72° C. were performed, followed by a final extension of 2min at 72° C. The amplified mixture was resolved by gel electrophoresis(2% agarose in TBE, containing 0.5 μg/mL of ethidium bromide) andvisualized under 254 nm UV illumination.

[0146] To assay the effectiveness of the enzymes tested, a PCR using thesame conditions as described above was run in parallel with theenzyme-treated samples. DNase I treatment sucessfully degraded all theDNA, as no amplifiable product was detected.

[0147] The detection limit of the RT-PCR of the DNase I-treated lysate(RNA only) was approximately 10 CFU per reaction. No signal was observedfrom samples treated with both RNase A and DNase I, thereby confirmingthat RNA was initially present in the lysate. Hence, one can concludethat RUCLANAP is thus a simple and effective method for the isolationfrom bacterial cells of total RNA that can successively be used for cDNAsynthesis.

Example 18

[0148] Extraction of genomic DNA from Cryptospordium parvum usingRUCLANAP. The universality of RUCLANAP was further demonstrated byobtaining crude genomic DNA from the parasite Cryptosporidium parvum.Oocytes were collected by centrifugation and resuspended in 50 μL of5×TE buffer. The basic RUCLANAP protocol (Example 1) was followed, usingthe yeast beads mix. One μL of a ten-fold dilution of the crudepreparation provided the template for specific amplification of theelongation factor 1 gene, using as primers SEQ ID NO. 798-800 and804-806 from our previous patent publication WO 01/23604. The expectedPCR products were detected by agarose gel electrophoresis.

Example 19

[0149] Extraction of genomic DNA from Mycobacterium smegmatis usingRUCLANAP. Another example of the universality of RUCLANAP is theisolation of crude genomic DNA from difficult to lyse Mycobacteriumsmegmatis. Robson et al. (U.S. Pat. No. 5,376,527) have shown thatcellular lysis of mycobacteria may be obtained by exposing the bacteriato a lysis-effective amount of heat, ranging from 2 to 20 minutes.Subsequently, DNA was further purified by phenol/chloroform extractionand ethanol-precipitated, which is much more cumbersome than theRUCLANAP method. Also, results showed that this method was not sensitiveenough for detection directly from clinical samples without prior growthenrichment. For some mycobacterial species, the heating steps includedin the PCR protocol were sufficient to release amplifiable target DNA,but this quick method was not universal for all Mycobacterium speciestested.

[0150] The basic RUCLANAP protocol was used with the universal lysis mix(Example 1). Starting from a 0.5 MacFarland suspension in PBS of M.smegmatis prepared from colonies, cells (50 μL) were collected bycentrifugation for 5 min at 21 000 g, the supernatant was discarded andthe pellet was resuspended in 50 μL of 5×TE buffer. After RUCLANAP, oneμL of serial ten-fold dilutions of the crude lysate provided thetemplates for specific amplification of the atpD gen , using a primerpair described in our previous patent publication WO 01/23604 (SEQ IDNO. 566 and 567). The unlysed cell suspension was also serially dilutedten-fold in PBS, and 100 μL from dilutions 10⁻⁵ and 10⁻⁶ were platedonto blood agar plates in triplicate to determine viable counts. One μLof each dilution of the unlysed cell suspension was also used astemplate in the PCR assay.

[0151] The unlysed cell suspension showed a count of 3.1×10⁴ CFU/μL. NoDNA amplification from all dilutions of the untreated cells suspensionwas detected by standard agarose gel electrophoresis, including thelowest dilution tested which contained 3.1×10⁴ CFU/PCR reaction. On theother hand, the RUCLANAP-treated M. smegmatis cells were detected with asensitivity limit of 3 CFU/PCR reaction. Treatment of the mycobacterialcells improved the detection by at least 10 000-fold. RUCLANAP is thus arapid and sensitive method to detect hard to lyse Mycobacteriumsmegmatis.

Example 20

[0152] RNA extraction from HIV-1 using RUCLANAP. A variation from thebasic RUCLANAP protocol was designed to extract RNA from type 1 humanimmunodeficiency virus (HIV-1) for comparison with the RNA extractionmethod of the NucliSens HIV QT assay from Organon Teknika. Thiscommercially available kit measures quantitatively HIV-1 RNA in humanserum and plasma. The RNA extraction process is derived from themethodology of Boom et al. (J. Clin. Microbiol., 1990, 28: 495-503);although effective, the assay is fastidious, time-consuming (a fewhours) and requires the use of the toxic substance guanidinethiocyanate.

[0153] Starting from 500 μL of human plasma with a known HIV-1 viralload, both RNA extraction methods were compared. The NucliSens HIV QTassay was performed as described in the manufacturer's instructions. ForRUCLANAP, 500 U of ribonuclease inhibitor RNasin® (Promega) were addedto the sample. After a 5 min centrifugation at 21 000 g, the pellet waswashed with 1 mL of 10 mM Tris-HCl, pH 8.0. After another centrifugationstep in the same conditions and removal of the supernatant, thefollowing were added: 0.2 g of beads (106 μm and finer, cat. no. G-4649,Sigma; presumably 75 μm-106 μm), 20 μL of RNA calibrators solution (seebelow), 10 μL of a 1% Triton X-100 solution, and 40 U of RNasin. Viruseswere disrupted in the FastPrep® FP-120 apparatus for 45 seconds at powerlevel 6. A final quick spin at 10 000 g for 2 min followed.

[0154] Amplification and detection of extracted RNA obtained from eithermethod was performed on the NucliSens Reader (Organon Teknika),according to the manufacturer's instructions. The technology used foramplification was NASBA (nucleic acid sequence-based amplification),which relies on the simultaneous activity of 3 enzymes: AMV-RT (AvianMyoblastosis Virus-Reverse Transcriptase), RNase H and T₇ RNApolymerase. Copies of the target RNA sequence and of the calibrators (3artificial control RNAs) are synthesized by phage T₇ polymerase via anintermediate DNA molecule harboring the double-stranded T₇ RNApolymerase promoter. Detection of products relies onelectrochemiluminescence (ECL) technology.

[0155] Using either viral RNA extraction method, similar levels ofrecovery were obtained, demonstrating the universality of the RUCLANAPmethod. Furthermore, RUCLANAP was more rapid, and simpler to use thanthe RNA extraction method of the NucliSens HIV QT assay. The universalmixture 150-212 μm: 710-1180 μm (4:1) could be used per se or adapted tolyse viruses (by adding smaller particles, namely in the 75-106 μmrange, by reducing the proportions of large beads or by providing aseparate composition of small particles only (about 150 μm or lower,namely 100 μm or lower)).

Example 21

[0156] Use of zirconia beads for RUCLANAP. The basic RUCLANAP protocolwas used with the universal lysis mix (Example 1), or with 0.05 g of 100μm zirconia/silica beads (cat. no. 11079101z, BioSpec). A negativecontrol, with no beads in the microtube, was also performed. Startingfrom a 0.5 MacFarland suspension in PBS of Staphylococcus aureus ATCC25923 prepared from colonies, cells (100 μL) were collected bycentrifugation for 5 min at 21 000 g, the supernatant was discarded andthe pellet was resuspended in 100 μL of 5×TE buffer. After RUCLANAP, oneμL of serial ten-fold dilutions (10⁰ to 10⁵) of the crude lysatesobtained in the three different conditions was used as template in theS. aureus-specific assay described by Martineau et al. (2000, J.Antimicrob. Chemother. 46: 527-534). Amplification was detected bystandard agarose gel electrophoresis. The unlysed cell suspension wasalso serially diluted ten-fold in PBS, and 100 μL from dilutions 10⁻⁵and 10⁻⁶ were plated onto blood agar plates in triplicate to determineCFU counts.

[0157] The unlysed cell suspension showed a count of 3.5×10⁴ CFU/μL. Thedetection limit for the negative control (no beads) was 350 CFU/PCRreaction. By contrast, the detection limit for RUCLANAP-treated S.aureus cells (using either the universal mix of glass beads orzirconia/silica beads) was 3.5 CFU/PCR reaction. Thus, RUCLANAPtreatment of the staphylococcal cells improved the nucleic aciddetection by at least 100-fold, and the method may be adapted todifferent types and sizes of beads.

[0158] Although the present invention has been described hereinabove byway of preferred embodiments thereof, it can be modified, withoutdeparting from the spirit and nature of the subject invention as definedin the appended claims. TABLE 1 Efficiency of DNA recovery using theRUCLANAP and 8 commercial kits. DNA EXTRACTION KITS AND METHODS^(a)Microbial RU- Dynabeads ® Fast RNA ™ Instagene ™ High Pure ™ PCRQIAamp ® Fast Gene species CLANAP DNA Direct ™ blue Kit Matrix TemplateKit NucliSens ™ Tissue Kit DNA ™ Kit Releaser ™^(b) E. coil ref −1 −1/2−1/2 0 −1/2 0 0 — S. aureus ref −3 −1/2 −2 −1 −2 −2 −4 — E. faecium ref−3 −1 −3 0 −4 −1 −3 — C. albicans ref −2 0 0 −1 −3 0 0 — Total^(c) 0 −9−2 −5,5 −2 −9,5 −3 −7 —

1. A method for releasing nucleic acids from microbial cells or viralparticles of a sample in preparation for nucleic acids testing (NAT)assays, comprising the steps of: a) disrupting the microbial cells orviral particles in a solution in the presence of a granulometricgradient formed by solid lysing particles having a diameter sizecomprised between 75 μm and 2000 μm, to release cellular componentsincluding nucleic acids into the solution, by applying a mechanicalforce sufficient to lyse the microbial cells or viral particles, so asto obtain a cellular or viral lysate; and b) controlling the presenceand/or activity of NAT assays inhibitors.
 2. A method as defined inclaim 1, wherein the granulometric gradient is formed by solid lysingparticles having a diameter size comprised between 75 μm and 1500 μm. 3.A method as defined in claim 1, wherein the granulometric gradient isformed by solid lysing particles having a diameter size comprisedbetween 75 μm and 1200 μm.
 4. A method as defined in claim 1, 2 or 3,wherein the step of controlling NAT assays inhibitors is performed byeither, a) removing the inhibitors out of the sample and/or the lysate;b) diluting the inhibitors in the sample and/or the lysate; c)inactivating the inhibitors in the sample and/or the lysate; or d) anycombination thereof.
 5. A method as defined in claim 4, wherein the stepof removing the inhibitors is a step of adsorbing the same out of thesample or the lysate.
 6. A method as defined in claim 4, wherein thestep of removing the inhibitors is a step of purifying the microbialcells or viral particles or the cellular components on a densitygradient.
 7. A method as defined in claim 4, wherein the step ofremoving the inhibitors is a step of or a repetition of steps ofcentrifuging and washing the microbial cells or viral particles or thecellular components.
 8. A method as defined in claim 4, wherein the stepof inactivating the inhibitors is a step of heating the sample or thelysate.
 9. The method of claim 8, wherein the step of heating comprisesa temperature from about 55° C. to about 100° C. and a time of heatingfrom about 15 seconds to about 15 minutes.
 10. The method of any one ofclaims 1 to 9, wherein said granulometric gradient is formed by solidlysing particles having a diameter size comprised between 75 μm and 1200μm.
 11. The method of any one of claims 1 to 9, wherein saidgranulometric gradient is discontinuons and formed by solid lysingparticles having a diameter size comprised between 150 and 212 μm andbetween 710 μm and 1180 μm.
 12. The method of any one of claims 1 to 11,wherein the particles are spherical.
 13. The method of any one of claims1 to 12, wherein said solid particles are composed of zirconium.
 14. Themethod of any one of claims 1 to 12, wherein said solid particles arecomposed of silica.
 15. The method of any one of claims 1 to 14, whereinthe ratio (w/w) of particles to sample is between about 4:1 and about1:2.
 16. The method of any one of claims 1 to 15, wherein said solutioncomprises a chelating agent.
 17. The method of claim 16, wherein saidchelating agent comprises 1 to 5 mM EDTA.
 18. The method of any one ofclaims 1 to 17, wherein said solution comprises a buffering agent. 19.The method of claim 18, wherein said buffered solution comprises 10 to50 mM Tris-HCl (pH 8.0).
 20. The method of any one of claims 1 to 19,wherein the sample comprises viruses, bacterial cells, yeast cells,fungal cells, parasitical cells, animal cells and/or human cells. 21.The method of claim 20, wherein the cells are yeast cells, and saidgranulometric gradient is formed by solid lysing particles having adiameter size comprised between 710 and 1180 μm.
 22. The method of claim20, wherein the cells are bacterial cells, and said granulometricgradient is formed by solid lysing particles having a diameter sizecomprised between 150 and 212 μm.
 23. The method of claim 20, whereinthe microorganisms are viruses, and said granulometric gradient isformed by solid lysing particles having a diameter size comprisedbetween 75 and 106 μm.
 24. The method of any one of claims 1 to 20,wherein said granulometric gradient is formed by solid lysing particleshaving at least two diameter size ranges, a first one comprised between150 and 212 μm and a second one comprised between 710 and 1180 μm. 25.The method of any one of claims 1 to 24, wherein step a) is performedwithin 5 minutes.
 26. A composition of matter for releasing nucleicacids from microbial cells or viral particles of a sample, whichcomprises a granulometric gradient formed by solid lysing particleshaving a diameter size comprised between 75 μm and 2000 μm.
 27. Thecomposition of claim 26, wherein said granulometric gradient is formedby solid lysing particles having a diameter size comprised between 75and 1500 μm.
 28. The composition of claim 26, wherein said granulometricgradient is formed by solid lysing particles having a diameter sizecomprised between 75 and 1200 μm.
 29. The composition of claim 26,wherein said granulometric gradient is discontinuous and formed by solidlysing particles having a diameter size comprised between 150 and 212 μmand between 710 μm and 1180 μm.
 30. The composition of any one of claims26 to 29, wherein said solid particles are composed of zirconium. 31.The composition of any one of claims 26 to 29, wherein said solidparticles are composed of silica.
 32. The composition of any one ofclaims 26 to 29, which comprises 0.02 g to 0.2 g of said solid particlesto be used with a volume of sample of 10 μL to 500 μL.
 33. Thecomposition of any one of claims 26 to 29, which further comprises achelating agent.
 34. The composition of claim 33, wherein, saidchelating agent is in an amount achieving a final concentration of 1 to5 mM EDTA.
 35. The composition of any one of claims 26 to 34, whichfurther comprises a buffering agent.
 36. The composition of claim 35,wherein said buffered solution comprises 10 to 50 mM Tris-HCl (pH 8.0).37. The composition of claim 26, wherein the cells are yeast cells, andsaid granulometric gradient is formed by solid lysing particles having adiameter size comprised between 710 and 1180 μm.
 38. The composition ofclaim 26, wherein the cell are bacterial cells, and said granulometricgradient is formed by solid lysing particles having a diameter sizecomprised between 150 and 212 μm.
 39. The composition of claim 26sforreleasing nucleic acids of viral particles, wherein said granulometricgradient is formed by solid lysing particles having a diameter sizecomprised between 75 and 106 μm.
 40. The composition of claim 26,wherein the granulometric gradient is formed by solid lysing particleshaving a diameter size comprised between 150 and 212 μm and between 710and 1180 μm.
 41. The method of claim 11 or 24, wherein the ratio (w/w)of particles having a diameter size comprised between 150 and 212 μm andbetween 710 and 1180 μm is 4:1.
 42. The composition of claim 29 or 40,wherein the ratio (w/w) of particles having a diameter size comprisedbetween 150 and 212 μm and between 710 and 1180 μm is 4:1.
 43. Thecomposition of any one of claims 26 to 42, which further comprises amaterial binding or adsorbing NAT assays inhibitors.
 44. The compositionof claim 43, wherein said material is activated carbon.
 45. The methodof any one of claims 5 and 10 to 25, wherein the step of adsorbing isperformed with activated carbon.